Collagen alpha1(I) gene (COL1A1) is repressed by RFX family

Collagen type I is composed of three polypeptide chains transcribed from two separate genes (COL1A1 and COL1A2) with different promoters requiring coordinate regulation. Our recent publications, centering on COL1A2 regulation, demonstrate that methylation in the first exon of COL1A2 at a regulatory factor for X box (RFX) site (at -1 to +20) occurs in human cancer cells and correlates with increased RFX1 binding and decreased collagen transcription (Sengupta, P. K., Erhlich, M., and Smith, B. D. (1999) J. Biol. Chem. 274, 36649-36655; Sengupta, S., Smith, E. M., Kim, K., Murnane, M. J., and Smith, B. D. (2003) Cancer Res. 63, 1789-1797). In normal cells, RFX5 complex along with major histocompatibility class II transactivator (CIITA) is induced by interferon-gamma to occupy this site and repress collagen transcription (Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2004) J. Biol. Chem. 279, 41319-41332). In this paper, we demonstrate that COL1A1 has an RFX consensus binding site surrounding the transcription start site (-11 to +10) that contains three methylation sites rather than one in the COL1A2 gene RFX binding site. RFX1 interacts weakly with the unmethylated COL1A1 site, and binds with higher affinity to the methylated site. RFX1 represses the unmethylated COL1A1 less efficiently than COL1A2. COL1A1 promoter activity is sensitive to DNA methylation and the COL1A1 gene is methylated in human cancer cells with coordinately decreased collagen expression. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (aza-dC) increases collagen gene expression with time in human cancer cells. On the other hand, RFX5 interacts with both collagen type I genes with a similar binding affinity and represses both promoters equally in transient transfections. Two dominant negative forms of RFX5 activate both collagen genes coordinately. Finally, CIITA RNA interference experiments indicate that CIITA induction is required for interferon gamma-mediated repression of both collagen type I genes.